1. Notes

2. Result Statistics

Figure 1. False discovery rate (FDR) curve. X axis is the number of peptide-spectrum matches (PSM) being kept. Y axis is the corresponding FDR.

Figure 2. PSM score distribution. (a) Distribution of PEAKS peptide score; (b) Scatterplot of PEAKS peptide score versus precursor mass error.

(a)

(b)

Figure 3. De novo result validation. Distribution of residue local confidence: (a) Residues in de novo sequences validated by confident database peptide assignment; (b) Residues in "de novo only" sequences.

(a)

(b)

Table 1. Statistics of data. # of MS scans 8667 # of MS/MS scans 173300 Table 2. Result filtration parameters. Peptide -10lgP ≥15 Peptide Ascore ≥0 Protein -10lgP ≥23 Proteins unique peptides ≥2 De novo ALC Score ≥50% Table 3. Statistics of filtered result. Peptide-Spectrum Matches 38906 Peptide sequences 19263 Protein groups 802 Proteins 802 Proteins (#Unique Peptides) 629 (>2); 173 (=2); 0 (=1); FDR (Peptide-Spectrum Matches) 20.2% FDR (Peptide Sequences) 36.6% FDR (Protein) 52.6% De Novo Only Spectra 25922

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Area AScore Methylation(KR) 14.02 KR 3574 127.53 1.51E5 168.11 Carbamidomethyl 57.02 C 3517 200.00 1.78E6 1000.00 Deamidation .98 NQ 2605 144.19 2.31E5 22.71 Oxidation 15.99 M 1990 160.67 8.38E5 1000.00 Methylation(others) 14.02 DEHST 1188 115.73 1.83E5 8.69 Phosphorylation 79.97 STY 786 97.99 2.18E5 0.00 Acetylation 42.01 N-term 731 84.44 8.18E4 1000.00 Pyro-glu from Q -17.03 N-term 674 142.35 1.97E5 1000.00 Dehydration -18.01 DSTY 611 82.01 5.56E4 8.14 Acetylation 42.01 K 464 72.22 1.99E4 50.00 Acetylation 42.01 Protein N-term 432 133.32 2.38E5 1000.00 Carbamylation 43.01 K,N-term 371 81.47   0.00 Dihydroxy 31.99 CFKPRWY 288 90.46 3.17E5 51.17 Sodium 21.98 DE,C-term 261 43.62 1.39E5 26.52 Formylation 27.99 K,N-term 245 53.68 2.3E5 51.38 Didehydro -2.02 STY,C-term 227 98.53   0.00 Amidation -.98 C-term 204 53.75   1000.00 Dimethylation(KR) 28.03 KR 194 84.05 5.05E5 61.82 Carboxylation 43.99 DKW 156 41.86   30.10 Biotin 226.08 K,N-term 131 42.35 1.14E5 60.72 Dethiomethyl -48.00 M 126 79.12 3.36E5 1000.00 Sulfation 79.96 STY 97 72.42   1000.00 Ethyl 28.03 DE,N-term,C-term 80 92.72 6.77E4 12.60 Pyroglutamic 13.98 P 79 53.92 2.47E5 61.69 Ammonia loss -17.03 N 77 94.70 1.27E5 71.03 Lysaminoadipicsealde -1.03 K 77 68.58   1000.00 Oxidation 15.99 HW 75 82.53 6.12E4 1000.00 Hexose 162.05 NSY 69 68.50 4.61E5 0.00 Deamidation .98 R 57 75.34 1.72E5 1000.00 Carboxylation 43.99 E 56 37.40 9.98E5 1000.00 Amidine 41.03 N-term 51 81.08 5.77E4 118.43 Carboxymethyl 58.01 C 48 94.95 4.88E5 47.09 Propionamide 71.04 K,N-term 39 54.94 1.81E5 63.99 Guanidination 42.02 K 38 96.82 1.2E5 200.29 MethylamineST 13.03 ST 31 67.83   15.73 Sulphone 31.99 M 28 71.48 8.59E5 1000.00 Methyl+Deamidated 15.00 NQ 27 60.83 6.74E4 20.75 Ubiquitin 114.04 KST 26 79.25 2.88E5 0.00 Diethylation 56.06 N-term 25 73.86 5.91E4 157.13 Cation:Li 6.01 DE,C-term 23 100.72 7.38E4 0.00 Pyro-glu from E -18.01 N-term 23 65.97 4.47E4 1000.00 O-Diisopropylphosphate 164.06 KSTY,N-term 15 59.03 4.3E4 21.19 Met->Aha -4.99 M 15 69.28 7.44E4 1000.00 HexNAc 203.08 N 13 23.34 7.77E4 1000.00 Oxidation 15.99 DKNPY 12 46.40 1.5E5 0.00 Acetyl 42.01 HST 12 32.67 3.17E5 37.82 Methylation(C-term) 14.02 C-term 12 47.95 5.43E5 1000.00 SMA 127.06 N-term 12 59.70 2.66E5 1000.00 EtOH 44.03 KR 12 32.43 3.89E4 0.00 Propionald+40 40.03 HK 11 58.34   0.00 glycidamide 87.03 K,N-term 10 51.94   84.20 Myristoylation 210.20 CK,N-term 10 30.03   34.72 Formylation 27.99 Protein N-term 10 30.39 4.42E5 1000.00 ISD_z+2_ion -15.01 N-term 10 59.61 3.21E5 1000.00 Propargylamine 37.03 DE,C-term 10 70.73   71.27 Phosphorylation 79.97 R 9 20.81   1000.00 Trifluoro 53.97 L 8 28.74 4.59E4 25.70 azole -20.03 S 8 39.02 9.18E4 110.48 Cation:K 37.96 DE,C-term 8 50.62 3.87E5 87.25 Oxolactone 13.98 W 8 45.04 8.54E4 1000.00 Kynurenin 3.99 W 7 81.30 1.77E5 1000.00 Iodination 125.90 HY 7 67.52 1.06E5 84.28 Aminoadipic 14.96 K 7 35.19 6.92E4 49.13 Carboxymethyl 58.01 K,N-term 7 39.80 1.68E5 26.08 ser_thr_DAET 87.05 ST 7 57.85 4.07E5 67.70 DSP 88.00 K,N-term 7 37.96 5.71E4 71.03 Ammonium 17.03 DE,C-term 7 48.18 1.04E5 3.36 Menadione-Q 170.04 K 7 49.50 1.23E5 1000.00 O-Ethylphosphate 108.00 KY,N-term 6 31.11 3.97E4 13.67 Arg2HPG 282.05 R 6 52.94 7.1E5 1000.00 B-methylthiol 45.99 DN 6 34.33 5.58E4 42.68 Pyridylacetyl 119.04 N-term 6 36.30 3.06E4 13.05 S-Eth 44.01 S 6 45.06 7.9E4 4.52 MDA54 54.01 K 6 28.94 1.28E5 0.00 Deoxy -15.99 DST 6 19.42 2.22E5 12.28 HNE 156.12 HK 6 74.57 1.9E5 167.97 Maleimide 97.02 K 6 29.11 2.25E5 1000.00 Argglutamicsealde -43.05 R 6 23.58 5.07E4 1000.00 Cation:Ni[II] 55.92 DE,C-term 5 37.11 1.69E4 6.08 Carboxyethyl 72.02 K 5 42.33 1.59E5 1000.00 Methylphosphonate 77.99 STY 5 52.78   4.08 PET 121.04 ST 5 34.63 5.85E4 14.04 Lipoyl 188.03 K 5 63.20 3.11E4 1000.00 Aminotyrosine 15.01 Y 5 56.11   23.15 G-H1 39.99 R 5 30.20 2.08E5 39.47 Phosphopropargyl 117.00 T 5 29.30 3.35E5 1000.00 O-Isopropylmethylphosphonate 120.03 SY 4 27.27 1.42E4 17.29 PEITC 163.05 K,N-term 4 57.82 9.01E4 10.11 Glu 129.04 E 4 40.52 6.15E4 1000.00 NDA 175.04 K,N-term 4 26.82   1000.00 O-Diethylphosphate 136.03 ST 4 36.49   7.77 Thio-phospho 95.94 ST 4 22.64 4.81E4 53.33 AEBS 183.04 KSY 4 29.69   72.73 Cation:Cu 61.92 D,C-term 4 37.68 2.09E5 24.24 Malonyl 86.00 S 4 28.64 1.12E5 1000.00 PyridoxalPhosphate 229.01 K 4 41.87 4.79E5 1000.00 PyMIC 134.05 N-term 4 32.52 4.85E4 1000.00 GPIanchor 123.01 C-term 4 17.53 2.33E4 1000.00

3. Experiment Control

Figure 4. Precursor mass error of peptide-spectrum matches (PSM) in filtered result. (a) Distribution of precursor mass error in ppm; (b) Scatterplot of precursor m/z versus precursor mass error in ppm.

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(b)

Table 5. Number of identified peptides in each sample by the number of missed cleavages

Missed Cleavages 0 1 2 3 4+ Atenuado 11648 5732 1557 326 0

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 0.07 Da Fragment Mass Error Tolerance: 0.07 Da Precursor Mass Search Type: monoisotopic Enzyme: Trypsin Max Missed Cleavages: 3 Non-specific Cleavage: none Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99   Acetylation (K): 42.01   Acetylation (Protein N-term): 42.01   Acetylation (N-term): 42.01   Amidation: -0.98   Beta-methylthiolation: 45.99   Biotinylation: 226.08   Carbamylation: 43.01   and 304 more... Max Variable PTM Per Peptide: 3 Database: Paracoccidioides_Pb18 Taxon: All Searched Entry: 7000 FDR Estimation: Enabled De novo score (ALC%) threshold: 15 Peptide hit threshold (-10logP): 30.0 Peaks run ID: 5 Different data refine parameters are used for this search:

Table 7. Instrument parameters. Fractions: AEV 2_3_RA2_01_1224.d, AEV_1_3_RA2_01_1232.d, AEV_3_ 3_RA3_01_1233.d Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: Quadrupole MS/MS Scan Mode: Time of Flight (TOF)